Splicing is an essential step in the expression of most eukaryotic genes. A dynamic RNA-protein complex, the spliceosome, containing 5 snRNAs and several dozen proteins, is assembled during the recognition of introns and exons in premessenger RNA. Rearrangements of RNA and protein within this complex establish the identity of phosphate bonds to be cleaved and joined for intron removal and exon ligation. Roles of snRNAs in recognition of each other and the premessenger RNA reactive sites have been demonstrated. The central questions posed by the observation that the spliceosome is a dynamic RNA machine are clear. How are snRNA rearrangements important for snRNA function in splicing? What is the biochemical nature of the factors that influence the initiation and control of these conformational changes? How are these events coordinated with regulation of the complex and its catalytic activity? Genetic approaches to this question with the budding yeast Saccharomyces cerevisiae have identified eight proteins that influence the activity of one of the snRNAs, U2. The recent realization that six of these proteins are the yeast homologs of subunits of the mammalian splicing factors SF3a and SF3b, the opportunity to study the function of a conserved set of splicing factors involved intimately with the function of spliceosomal RNAs has been realized. The proposed experiments will make use of this opportunity by focusing on completing the description of yeast SF3b subunit composition, primary structure and subunit interactions using genetic and molecular approaches based on the analysis of the CUS1 and HSH49 proteins. Two other conserved proteins that influence U2 snRNP activity in the splicing pathway will also be studied with experiments that will: (1) determine the composition and subunit interactions within complexes that contain CUS1 and HSH49, as well as identify relationships between such complexes and other splicing factors, (2) identify the role of CUS2 in splicing with respect to possible functional similarities between CUS2 and human Tat-SF1, and (3) analyze the structure and function of the DEAD-box protein PRP5 in splicing with attention to its interaction with conserved U2 snRNP proteins and its role in U2 snRNA dynamics. These studies will provide the necessary structural framework in which to develop a mechanistic understanding of the roles of proteins that interact with snRNAs in the splicing pathway.